Method and computer program product for distinguishing and sorting seeds containing a genetic element of interest

ABSTRACT

A method and computer program product for distinguishing and sort seeds containing a genetic element of interest from a bulk sample. In various embodiments, the present invention comprises associating a marker with at least some of the seeds containing a genetic element of interest of the bulk sample, exciting the seeds using an electromagnetic energy emitting device, evaluating at least some of the seeds of the bulk sample for the presence or absence of the marker using an evaluating device configured to excite a majority of the surface area of the seeds, and sorting the seeds containing a genetic element of interest based on the presence or absence of the marker. In various other embodiments, the method and computer program product may comprise associating a red fluorescent protein marker with at least some of the seeds containing a genetic element of interest of the bulk sample, evaluating at least some of the seeds of the bulk sample for the presence of the red fluorescent protein marker using an evaluating device, and sorting the seeds containing a genetic element of interest based on the presence of the red fluorescent protein marker. In some embodiments, the red fluorescent protein marker is discernable when excited by a certain energy and the evaluating step comprises exciting the seeds containing a genetic element of interest with the certain energy detecting an emission resulting at least in part from the exciting step.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority from U.S. ProvisionalApplication No. 60/913,562, filed Apr. 24, 2007, which is herebyincorporated herein in its entirety by reference.

FIELD OF THE INVENTION

The various embodiments of the present invention relate generally to amethod and computer program product for distinguishing and sorting seedsthat contain genetic elements of interest from seeds that do not. Morespecifically, embodiments of the present invention are capable ofdistinguishing and sorting the seeds containing the genetic elements ofinterest by detecting the presence of one or more discernable markersthat are linked to one or more corresponding genetic elements ofinterest.

BACKGROUND OF THE INVENTION

Genetic engineering involves inserting new genetic information intoexisting cells of an organism to create a new genotype in order tomodify the organism for the purpose of changing one or more of itscharacteristics. A living organism may be genetically engineered for avariety of reasons, including incorporating novel and/or beneficialtraits into the organism. With regard to crop plants, this may includegenetically engineering seeds so that plants that grow from the seedsinclude one or more beneficial traits. Such genetic engineering mayinclude, but is not limited to, inserting genes encoding a discernablemarker or genes conferring resistance to herbicidal compounds. Theprogeny of individual genetically modified organisms may also contain agenetic element of interest such that researchers may wish to segregateand/or identify organisms (including seeds) that contain such geneticelements of interest from a bulk sample.

Conventional genetic techniques may also be used to combine traits fromat least two organisms to produce novel and/or beneficial traits in a“genetic cross” and/or hybrid organism.

It is often difficult to determine whether a particular seed contains agenetic element of interest, especially when mixed with seeds that havenot been so modified. Using previous techniques, a particular seed wouldhave to be germinated and then the resulting plant sampled to determinewhether the seed might contain the genetic element of interest.Alternatively, the seed itself would have to be sampled.

There are advantages, however, in distinguishing a set of seedscontaining a genetic element of interest before the respective plantsare germinated, especially in the plant research discipline, whereresearch plot space, personnel for sorting, and time are often limited.In maize and other wind-pollinated crops, plants are frequentlyhand-pollinated, wherein pollen is manually transferred from the tasselto the silk, and the silk is covered by a shoot bag to preventpollinated by other plants. This hand pollination process requiressignificant labor and a tassel bag and shoot bag. The prior eliminationof undesired plants (i.e. plants that do not contain a genetic elementof interest) by sorting seed may eliminate the need for this labor andtime-intensive activity. Additionally, researchers may be interested inthe yield potential of the plants germinated from seeds containing thegenetic element of interest. Thus, it is important that astatistically-significant number of plants germinate from seedscontaining the genetic element of interest in a defined space (i.e. aresearch plot). Furthermore, researchers desire accuracy and oftencannot afford to wait or guess to determine whether plants contain aparticular genetic element of interest. Additionally, seed and/or seedtissue sampling is a delicate art. For example, if too much tissue isremoved from a particular seed for sampling, there is a risk that theseed may not germinate or produce a viable plant.

Some processes have been disclosed for visualizing green fluorescentprotein (GFP) expression in transgenic plants in order to selecttransgenic seeds as described, for example, in U.S. Pat. No. 6,947,144to Kim et al. In particular, the Kim reference describes a system forseparating seeds transformed with a green fluorescent protein (GFP) thatincludes a light source 5′ filtered through a bandpass filter 6′. Thelight source 5′ is positioned above and at a 45° angle from plantsamples traveling on a conveyor belt 3′. A CCD camera 9′ is alsopositioned above the conveyor belt 3′, directly above the examined plantsample 4′. The CCD camera 9′ detects light generated from a portion ofthe surface area of the plant sample through a filter 7′. See, e.g., theKim reference, FIG. 9.

However, the system disclosed by the Kim reference suffers from severalinsufficiencies. For example, there are a number of different traits,including the expression of fluorescent proteins, for which the accuratemeasurement of a seed in more than a portion of the surface area of theseed may be important. In some instances, the expression of afluorescent protein may not be uniform across entire surface area of aseed. Thus, if a localized area of the seed expresses the fluorescentprotein and that area is facing downward (such as against the conveyorbelt in the Kim reference) the CCD camera will not detect thefluorescent protein and the seed will not be correctly sorted.

In addition, the use of GFP for identification and/or selection oftransgenic plant material presents several technical challenges. Firstof all, the excitation and emission wavelengths for GFP visualizationare on the fringes of the visible spectrum, and are therefore not easilyvisible to the eye (or to many conventional visualization systems) usingnormal “white” light that is present in conventional research and/ormanufacturing environments. Furthermore, it has been noted that GFP mayresult in protein aggregation in vivo in plant material that has beentagged with GFP. It is well-known that unchecked protein aggregation maynot only produce adverse effects in seed product, but may also produceunwanted environmental effects if GFP-tagged seeds are introduced intoan agricultural environment that interacts with external ecologicalsystems (such as forests and/or wetlands adjacent to an agriculturalresearch plot).

As a result, there is a need for a method and computer program productfor distinguishing seeds that contain a marker associated with aselected genotype from a bulk sample of seeds. The method and computerprogram product should permit a bulk sample to be sorted based on thepresence of the marker or the absence of the marker, and should providea level of convenience, accuracy, speed and environmental safety that isnot available using conventional techniques. Additionally, the methodand computer program product should provide the ability to accuratelysort a bulk sample of seeds based on the presence or absence of markersassociated with a selected genotype using commercially-availablehigh-speed sorting equipment with minimal modifications.

SUMMARY OF THE INVENTION

The embodiments of the present invention satisfy the needs listed aboveand provide other advantages as described below. Some embodiments of thepresent invention may include a method for distinguishing seedscontaining a genetic element of interest from a bulk sample byassociating a discernable marker with at least some of the seedscontaining a genetic element of interest of the bulk sample, evaluatingat least some of the seeds of the bulk sample for the presence orabsence of the marker using an evaluating device, and sorting the seedscontaining a genetic element of interest based on the presence orabsence of the marker.

In some embodiments the seeds are excited using an electromagneticenergy emitting device configured to excite a majority of the surfacearea of the seeds. In some embodiments, exciting the seeds using anelectromagnetic energy emitting device comprises exciting the seedsusing at least one illuminating device configured to illuminate amajority of the surface area of the seeds. In some embodiments, excitingthe seeds using at least one illuminating device further comprises usingtwo or more illuminating devices arranged to collectively illuminate amajority of the surface area of the seeds. In some embodiments,evaluating at least some of the seeds of the bulk comprises evaluatingat least some of the seeds of the bulk sample using at least one imagesensing device arranged to receive emissions from a majority of thesurface area of the seeds. In some embodiments, evaluating at least someof the seeds of the bulk sample further comprises using two or moreimage sensing devices arranged to collectively receive emissions from amajority of the surface area of the seeds.

Other embodiments of the present invention may include a method andcomputer program product for distinguishing seeds containing a geneticelement of interest from a bulk sample by associating a discernable redfluorescent protein (RFP) marker with at least some of the seedscontaining a genetic element of interest of the bulk sample, evaluatingat least some of the seeds of the bulk sample for the presence orabsence of the RFP marker using an evaluating device, and sorting theseeds containing a genetic element of interest based on the presence orabsence of the RFP marker.

In some embodiments, the RFP marker is discernable when excited by acertain energy having a wavelength ranging from substantially about 500nm to substantially about 580 nm. In some such embodiments, the certainenergy may have a peak at substantially about 550 nm. In otherembodiments, the evaluating step comprises exciting the seeds containinga genetic element of interest with the certain energy; and detecting anemission resulting at least in part from the exciting step. In some suchembodiments, the resulting emission may have a wavelength ranging fromsubstantially about 500 nm to substantially about 600 nm. Furthermore,the emission may have a peak at substantially about 580 nm. In someembodiments, the evaluating device may further comprise at least onefilter disposed substantially between the image sensing device and theseeds containing a genetic element of interest. The filter may beconfigured for passing the emission from the red fluorescent proteinmarker to the image sensing device.

Some embodiments may further comprise associating a plurality ofsupplemental markers with at least some of the seeds containing acorresponding plurality of additional genetic elements of interest ofthe bulk sample. Such embodiments may further comprise steps forevaluating at least one of the seeds of the bulk sample for the presenceor absence of the plurality of supplemental markers using the evaluatingdevice, and sorting the seeds containing the plurality of additionalgenetic elements of interest based on the presence or absence of the aplurality of supplemental markers. In such embodiments, the supplementalmarkers may include, but are not limited to: yellow fluorescentproteins; yellow/orange fluorescent proteins; orange fluorescentproteins; orange/red fluorescent proteins; red/orange fluorescentproteins; red fluorescent proteins; cyan fluorescent proteins; andcombinations of such supplemental markers.

In some embodiments, the evaluating device may be a seed color sorter.In other embodiments, the image sensing device may be a charge-coupleddevice (CCD device) or a complementary metal oxide semiconductor (CMOS)device. In other embodiments, the evaluating step comprises evaluatingseeds for the presence of a RFP marker using at least one image sensingdevice configured to differentiate between a range of normal seedemissions and the discernable RFP marker. In other embodiments, thesorting step may comprise dispensing the sorted individual seeds into atleast one container, the container including only seeds having the RFPmarker present or only seeds in which the RFP marker is absent.

Other embodiments further comprise steps for singulating individualseeds from the bulk sample using a singulating device. In someembodiments, the singulating step comprises singulating seeds using aplurality of elongate hollow structures operatively connected to avacuum source. In other embodiments, the singulating step comprisesreceiving the bulk sample in a hopper and vibrating the hopper so as topropel individual seeds along an inclined spiral ramp and through a gapconfigured to permit the passage of one seed at a time. In otherembodiments, the singulating step comprises receiving the bulk sample ina hopper, feeding seeds from the hopper to a rotatable disk, androtating the rotatable disk so as to distribute the seeds to a peripheryof the rotatable disk by application of centrifugal force. In otherembodiments, the singulating step comprises receiving the bulk sample ina hopper, vibrating the bulk sample such that seeds tend to move towardsan opening in the hopper, distributing seeds from the opening in thehopper to a sloped device that includes at least one groove such that aline of individual seeds is formed in the groove, and depositingindividual seeds by gravity force from the sloped device onto aconveying device.

Thus the various embodiments of distinguishing seeds containing agenetic element of interest of the present invention provide manyadvantages that may include, but are not limited to: providing a methodand computer program product capable of permitting a bulk sample ofseeds to be sorted based on the presence or absence of a marker ormultiple RFP markers, associated with a selected genotype, or multipleselected genotypes. The present invention provides a level ofconvenience, speed, accuracy, and environmental safety that is notavailable using conventional techniques. Additionally, the presentinvention provides additional processing of the sorted seeds, includingdispensing the sorted seeds into containers.

These advantages, and others that will be evident to those skilled inthe art, are provided in the method and computer program product of thepresent invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus described the invention in general terms, reference will nowbe made to the accompanying drawings, which are not necessarily drawn toscale, and wherein:

FIG. 1 shows a flow chart of a method, according to one embodiment ofthe present invention, including steps for associating a marker withseeds containing a genetic element of interest, evaluating the seeds forthe presence of the marker, and sorting the seeds based on the presenceor absence of the marker;

FIG. 2 shows a non-limiting schematic of a seed sorter device used todistinguish seeds containing a genetic element of interest in accordancewith one embodiment of the present invention;

FIG. 3 shows a non-limiting schematic of a system incorporating a vacuumdrum singulating device for singulating seeds prior to evaluating andseparating seeds based on the presence of a marker in accordance withone embodiment of the present invention;

FIG. 4 shows a non-limiting schematic of a system incorporating a hopperhaving an inclined ramp for singulating seeds prior to evaluating andseparating seeds based on the presence of a marker in accordance withone embodiment of the present invention;

FIG. 5 shows a non-limiting schematic of a system incorporating arotatable disk for singulating seeds prior to evaluating and separatingseeds based on the presence of a marker in accordance with oneembodiment of the present invention; and

FIG. 6 shows a non-limiting schematic of a system incorporating aninclined ramp having a groove feature for singulating seeds prior toevaluating and separating seeds based on the presence of a marker inaccordance with one embodiment of the present invention;

FIG. 7 shows a non-limiting block diagram of an exemplary electronicdevice configured to distinguish genetically transferred seeds from abulk sample of exemplary embodiments of the present invention;

FIG. 8 shows a non-limiting schematic of a seed sorter device used todistinguish seeds containing a genetic element of interest in accordancewith one embodiment of the present invention;

FIG. 9 shows a non-limiting schematic of a system incorporating a vacuumdrum singulating device and a pair of illuminating devices in accordancewith one embodiment of the present invention; and

FIG. 10 shows a non-limiting schematic of a seed sorter deviceincorporating a pair of illuminating devices and a pair of evaluatingdevices used to distinguish seeds containing a genetic element ofinterest in accordance with one embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, this invention may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Under current methods, it is difficult to distinguish, prior togermination, seeds that contain a genetic element of interest from seedsthat do not contain such elements. To address this concern, variousembodiments of the present invention provide a method and computerprogram product that sorts seeds based at least in part on therelationship between a discernable marker and one or more seedscontaining a genetic element of interest. For the purposes of thecurrent specification and the appended claims, the term “discernable”refers to an ability to recognize a marker when excited by certainenergy. In various embodiments, this may include the visible portion ofthe electromagnetic spectrum, as well as Ultraviolet and Infraredspectrums.

FIG. 1 shows a flowchart illustrating a method 10 for distinguishingseeds containing a genetic element of interest from a bulk sample. Instep 12, a marker is associated with at least a portion of seedscontaining a genetic element of interest from the bulk sample. Invarious embodiments, associating a marker with at least a portion ofseeds containing a genetic element of interest may comprise a variety oftechniques used to mark a desired genotype. In an exemplary embodiment,associating a marker with at least a portion of seeds containing agenetic element of interest comprises attaching DNA sequences that codefor red fluorescent protein (RFP) to a desired trait DNA sequence thatis inserted into a seed or a group of seeds. It should be noted thatalthough the exemplary embodiment described below associates an RFPmarker with a desired trait, in other embodiments multiple fluorescentprotein (FP) markers may be associated with a desired trait DNA sequenceor multiple desired trait DNA sequences in the same seed. Suchadditional FP markers may include, but are not limited to: yellow;yellow/orange; orange; orange/red, red/orange; red (including the RFPdescribed herein); and cyan. In other embodiments, the marker maycomprise phenotypic markers such as β-galactosidase and fluorescentproteins such as cyan florescent protein (CYP) (Bolte et al. (2004) J.Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol129:913-42), and yellow florescent protein (PhiYFP™ from Evrogen, see,Bolte et al. (2004) J. Cell Science 117:943-54). For additionalselectable markers, see generally, Yarranton (1992) Curr. Opin. Biotech.3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA89:6314-6318; Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol.Microbiol. 6:2419-2422; Barkley et al. (1980) in The Operon, pp.177-220; Hu et al. (1987) Cell 48:555-566; Brown et al. (1987) Cell49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al. (1989)Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc.Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al. (1990) Science248:480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg;Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow etal. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc.Natl. Acad. Sci. USA 89:3952-3956; Baim et al. (1991) Proc. Natl. Acad.Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res.19:4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol.10:143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother.35:1591-1595; Kleinschnidt et al. (1988) Biochemistry 27:1094-1104;Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al.(1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992)Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbookof Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill etal. (1988) Nature 334:721-724. Such disclosures are herein incorporatedby reference. In other embodiments, the marker may also comprise aselectable marker gene for the selection of transformed cells.

The above lists are not meant to be limiting. Any desired trait andmarker may be used in the present invention.

In various embodiments, the desired trait DNA sequence may correspondsubstantially to any trait desired to be inserted into a plant genome.

In one exemplary embodiment, the marker associated with at least aportion of seeds containing a genetic element of interest from the bulksample in step 12 may comprise a RFP marker such as the DsRed and/orDsRed2 RFP markers from the Living Colors™ line of novel fluorescentproteins (NFP) commercially available from Clontech Laboratories, Inc.of Mountain View, Calif. The use of RFP as the marker may provideseveral advantages over other fluorescent protein markers (such as greenfluorescent protein (GFP)). For example, unlike GFP, RFP excitation andemission wavelengths are closer to a center of the visible range of theelectromagnetic spectrum. Therefore, RFP-expressing seed may be visibleto the eye (and unassisted and/or unmodified cameras or other sensordevices) in ‘normal’ ambient white light. Thus, seed sorting (step 16,for example) may be accomplished in such embodiments without the needfor specialized UV light sources and/or detectors. Furthermore, manycommercially available and industry-proven high-speed sorting devicesoperate most effectively in the visible range of the electromagneticspectrum. Again as above, since RFP excitation and emission wavelengthsare predominantly centered in the visible range of the electromagneticspectrum, sorting (step 16) may be accomplished using existing andreadily-available equipment as an add-on to the current use for morestandard quality sorting procedures. Furthermore, RFP (and specificallyDsRed2) markers are optimized for minimal protein aggregation, such thatthe marker may avoid issues with in vivo protein aggregation that mayaffect product performance (i.e. seed viability and/or yield) as well asproduce potentially negative environmental effects that run counter toprinciples of product stewardship.

Step 12 may comprise, in some embodiments, associating the RFP markerwith one or more desired trait DNA sequences that may be inserted into aseed or a group of seeds. For example, genetically engineered plantsand/or seeds may contain beneficial traits of interest such as one ormore genes expressing peptides with pesticidal and/or insecticidalactivity, such as Bt toxic proteins (described in, for example, U.S.Pat. Nos. 5,277,905; 5,366,892; 5,747,450; 5,723,756; 5,859,336;5,593,881; 5,625,136; 5,689,052; 5,691,308; 5,188,960; 6,180,774;6,023,013; 6,218,188; 6,342,660; 6,114,608; and 7,030,295; USPublication Nos: US20040199939 and US20060085870; WO2004086868; andGeiser et al. (1986) Gene 48:109) and Bt crystal proteins of the Cry34and Cry35 classes (see, e.g., Schnepf et al. (2005) Appl. Environ.Microbiol. 71:1765-1774), and vegetative insecticidal proteins (forexample, members of the VIP1, VIP2, or VIP3 classes, see, for example,U.S. Pat. Nos. 5,849,870; 5,877,012; 5,889,174; 5,990,383; 6,107,279;6,137,033; 6,291,156; 6,429,360; as well as U.S. Pat. App. PublicationNos: US20050210545; US20040133942; and US20020078473), lectins (VanDamme et al. (1994) Plant Mol. Biol. 24:825, pentin (described in U.S.Pat. No. 5,981,722), lipases (lipid acyl hydrolases, see, e.g., thosedisclosed in U.S. Pat. Nos. 6,657,046 and 5,743,477), cholesteroloxidases from Streptomyces, and pesticidal proteins derived fromXenorhabdus and Photorhabdus bacteria species, Bacillus laterosporusspecies, and Bacillus sphaericus species, and the like. Alsocontemplated is the use of chimeric (hybrid) toxins (see, e.g., Bosch etal. (1994) Bio/Technology 12:915-918).

Genetically engineered plants and/or seeds thereof can also contain oneor more genes with a variety of desired trait combinations including,but not limited to, traits desirable for animal feed such as high oilgenes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.,hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and5,703,049); barley high lysine (Williamson et al. (1987) Eur. J.Biochem. 165:99-106; and WO 98/20122) and high methionine proteins(Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988)Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12:123));increased digestibility (e.g., modified storage proteins (U.S. Pat. No.6,858,778); and thioredoxins (U.S. Pat. No. 7,009,087)). Such plants canalso contain one or more genes resulting in traits desirable for diseaseresistance (e.g., fumonisin detoxification genes (U.S. Pat. No.5,792,931); avirulence and disease resistance genes (Jones et al. (1994)Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al.(1994) Cell 78:1089).

Genetically modified plants may further contain one or more genesencoding one or more forms of herbicide resistance, for example, toglyphosate-N-(phosphonomethyl) glycine (including the isopropylaminesalt form of such herbicide). Exemplary herbicide resistance genesinclude glyphosate N-acetyltransferase (GAT) and5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), including thosedisclosed in U.S. Pat. Application Publication Nos: US20040082770, alsoWO02/36782 and WO03/092360). Herbicide resistance genes generally codefor a modified target protein insensitive to the herbicide or for anenzyme that degrades or detoxifies the herbicide in the plant before itcan act. See, e.g., DeBlock et al. (1987) EMBO J. 6:2513; DeBlock et al.(1989) Plant Physiol. 91:691; Fromm et al. (1990) BioTechnology 8:833;Gordon-Kamm et al. (1990) Plant Cell 2:603; and Frisch et al. (1995)Plant Mol. Biol. 27:405-9. For example, resistance to glyphosate orsulfonylurea herbicides has been obtained using genes coding for themutant target enzymes, EPSPS and acetolactate synthase (ALS). Resistanceto glufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate(2,4-D) have been obtained by using bacterial genes encodingphosphinothricin acetyltransferase, a nitrilase, or a2,4-dichlorophenoxyacetate monooxygenase, which detoxify the respectiveherbicides. Also contemplated are inhibitors of glutamine synthase suchas phosphinothricin or basta (e.g., bar gene).

Transgenic plants and/or seeds may also contain one or more genesresulting in traits desirable for processing or process products such asmodified oils (e.g., fatty acid desaturase genes (U.S. Pat. Nos.5,952,544; 6,372,965)); modified starches (e.g., ADPG pyrophosphorylases(AGPase), starch synthases (SS), starch branching enzymes (SBE), andstarch debranching enzymes (SDBE)); and polymers or bioplastics (e.g.,U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyratesynthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J.Bacteriol. 170:5837-5847)). Such plants could also contain one or moregenes providing agronomic traits such as male sterility (e.g., see U.S.Pat. No. 5,583,210), stalk strength, flowering time, or transformationtechnology traits such as cell cycle regulation or gene targeting (e.g.,WO 99/61619, U.S. Pat. Nos. 6,518,487 and 6,187,994).

In step 14, the seeds from the bulk sample are evaluated for thepresence or absence of the RFP marker. In various embodiments, this step14 may comprise visually determining whether the RFP marker is presentor absent in a seed or a group of seeds. In an exemplary embodiment,step 14 comprises exciting seeds from the bulk sample with a certainenergy and then determining whether the RFP marker is present. Thecertain energy used to illuminate the seeds (and/or excite the RFPmarkers that may be present therein) may have a wavelength substantiallywithin the visible electromagnetic spectrum (i.e. a wavelength rangingfrom substantially about 500 nm to substantially about 580 nm). In someembodiments the certain energy emitted as part of step 14 may exhibit apeak at substantially about 550 nm. In some embodiments, step 14 mayfurther comprise detecting an emission resulting at least in part fromthe exciting step, wherein the emission may have a wavelength rangingfrom substantially about 500 nm to substantially about 600 nm. In someembodiments, the emission resulting from step 14 may exhibit a peak atsubstantially about 580 nm.

Various evaluating devices may be used for the determination performedin step 14. For example, evaluating devices may include, but are notlimited to, commercially available optical seed sorters, such as aScanMaster II seed sorter manufactured by Satake-USA. Although in normalusage, seed sorters may be used to evaluate and sort out contaminantssuch as rocks, glass, soil, damaged food items, mold, and other foreignmaterial from a bulk sample of food items, an exemplary embodiment ofthe present invention modifies a seed sorter to evaluate and sort seedsexpressing a fluorescent protein that is attached to a desired trait DNAsequence that has been inserted into a seed or a group of seeds in thebulk sample.

As shown generally in FIG. 8, such optical seed sorters may include aspecialized vision system 54 which, in some embodiments, may comprise:(1) an illuminating device 54 a (such as a bulb, for example) that emitselectromagnetic energy at a particular frequency (characterizing the“certain energy” described herein) chosen to illuminate and thereby“excite” the RFP marker, and (2) a filter 54 b used that may be used tofilter the energy emitted (i.e. “the emission”) from the RFP-taggedseeds so that a camera 54 c (or other image sensing device) of thevision system may discern seeds expressing the RFP marker.

To accomplish this task, in one method embodiment of the presentinvention, the evaluating step 14 may comprise evaluating seeds for thepresence of the RFP marker using at least one image sensing device 54 cconfigured to differentiate between a range of normal seed emissions andan emission from the RFP marker. As described herein, such an imagesensing device 54 c may comprise a component of a commercially availablecolor sorter device. Furthermore, in some embodiments, the image sensingdevice 54 c may comprise a CCD device and/or a CMOS device configuredfor detecting the emission from RFP-tagged transgenic and/or otherwisegenetically-modified seeds.

As shown in FIG. 8, in some embodiments, the evaluating device employedas part of the evaluating step 14 may further comprise at least onefilter 54 b disposed substantially between an image sensing device 54 c(such as a CCD) and the seeds containing a genetic element of interest.The at least one filter 54 b may be configured for passing the emissionfrom the red fluorescent protein marker to the image sensing device 54c. For example, in some embodiments, the filter 54 b may comprise aband-pass filter configured for passing electromagnetic energy having awavelength that is substantially equivalent to the targeted emissionwavelength (i.e. the emission wavelength of energy emitted from anilluminated and subsequently excited RFP marker (such as DsRed2, forexample). The emission may then be detected by the image sensing device54 c that may be further configured for translating the emission intosubstantially “white” light. Thus, the image sensing device may assignsubstantially binary values to each seed based on the presence orabsence of the RFP marker wherein seeds containing a genetic element ofinterest (tagged with the relatively “bright” RFP marker) are marked“positive” (and thereby deflected and/or other wise directed into one ormore “+” containers 56. Seeds that do not contain a genetic element ofinterest, or other particulate debris (which may be translated into a“dark” or “negative” result) may be dropped and/or otherwise directedinto one or more “−” containers 58. As shown in FIG. 8, the imagesensing device 54 c (or other component of a vision system 54 may be incommunication with a sorting device 55 (which may comprise, for example,a valve device and/or compressed air jet device) configured fordirecting the “positive” (i.e. seeds containing a genetic element ofinterest (which the image sensing device 54 c may detect as “white” or“bright” seeds)) into one or more “+” containers 56 in response to abinary positive or “1” signal received from the image sensing device 54c or other processing component of the vision system 54. The sortingdevice 55 may also be configured for directing the “negative” (i.e.seeds that do not contain a genetic element of interest or particulatedebris (which the image sensing device 54 c may detect as “dark” seeds))into one or more “−” containers 58 in response to a binary negative or“0” signal received from the image sensing device 54 c or otherprocessing component of the vision system 54. While the system 50 shownin FIG. 8 is shown oriented in a substantially vertical orientation(such that individual seeds pass through the vision system components 54a, 54 b, 54 c in response to gravity forces) in should be understoodthat the system 50 may also be oriented substantially horizontally andmay comprise one or more pressurized pneumatic tubes and/or conveyancepathways configured for directing individual seeds from a sample pastthe various vision system components 54 a, 54 b, 54 c and subsequentlyto a sorting device 55 that may be configured for transferring the seedscontaining a genetic element of interest into corresponding “+”containers 56 and for transferring the seeds not containing the geneticelement of interest into corresponding “−” containers 58 in response tosignals received from one or more vision system 54 components and/orcontrollers.

As described herein, some embodiments of the present invention mayutilize a plurality of supplemental markers (such as a variety offluorescent protein markers) to highlight the presence and/or absence ofa corresponding plurality of additional genetic elements of interest inthe seeds of the bulk sample. For example, as described herein, variousembodiments of the present invention may utilize supplemental markersthat may include, but are not limited to: yellow fluorescent proteins;yellow/orange fluorescent proteins; orange fluorescent proteins;orange/red fluorescent proteins; red/orange fluorescent proteins; redfluorescent proteins; cyan fluorescent proteins; and combinations ofsuch supplemental markers. Referring generally to FIG. 1, suchembodiments may comprise step 12 for associating one or more of aplurality of supplemental markers with at least some of the seedscontaining a corresponding plurality of additional genetic elements ofinterest of the bulk sample. Some such embodiments may further comprisestep 14 for evaluating at least one of the seeds of the bulk sample forthe presence or absence of the plurality of supplemental markers usingthe evaluating device and subsequently, step 16 for sorting the seedscontaining the plurality of additional genetic elements of interestbased on the presence or absence of the a plurality of supplementalmarkers.

In some such embodiments, the evaluating step 14 may comprise evaluatingseeds for the presence of at least one of the plurality of supplementalmarkers using at least one image sensing device 54 configured todifferentiate between a range of normal seed emissions and at least oneemission from one or more of the plurality of supplemental markers. Asdescribed herein, in some embodiments, the image sensing device 54 maycomprise a CCD device or a CMOS device. As shown generally in FIG. 8,the evaluating device used to perform the evaluating step 14 maycomprise a specialized vision system 54 which, in some embodiments, maycomprise: (1) an illuminating device 54 a (such as a bulb, for example)that emits electromagnetic energy at a particular frequency(characterizing the “certain energy” described herein) chosen toilluminate and thereby “excite” the RFP marker, and (2) a filter 54 bused that may be used to filter the energy emitted (i.e. “the emission”)from the RFP-tagged seeds so that a camera 54 c (or other image sensingdevice) of the vision system may discern seeds expressing the RFPmarker. In some embodiments wherein a plurality of supplemental markers(which may each exhibit a characteristic emission frequency, forexample) are used to “tag” a corresponding plurality of additionalgenetic elements that may be present in the seeds, the filter 54 b maycomprise at least one tunable filter disposed substantially between theimage sensing device 54 c and the seeds containing a genetic element ofinterest. The at least one tunable filter 54 b may be configured forselectively passing the at least one emission from one or more of theplurality of supplemental markers to the image sensing device 54 c. Insuch embodiments, the tunable filter 54 c may allow the evaluatingdevice to selectively sort for a plurality of different genetic elementsof interest that may be present in the seed sample. In one exemplaryembodiment, the filter device 54 c may comprise a VariSpec™ LiquidCrystal Tunable Filter (LCTF) commercially available from CRI, Inc. ofWoburn, Mass. In some such embodiments, the filter 54 b may be “tuned”to sequentially sense and sort seeds based on “positive” emissions fromseeds having one particular genetic element of interest (thatcorresponds, for example, to one of a plurality of supplemental markersand/or the base RFP) during a sequence of passes through the evaluatingdevice 54.

FIG. 2 shows a schematic representation of another exemplary system 50that may be used to distinguish seeds containing a genetic element ofinterest from a bulk sample of seeds in accordance with one exemplaryembodiment. In the exemplary embodiment, the system 50 is a modifiedcommercially available seed sorter. A bulk sample A of seeds, at leastsome of which have been genetically transformed by inserting a desiredtrait DNA sequence with an attached DNA sequence that codes forfluorescent proteins, are loaded into a hopper 52. The hopper 52 isconfigured to funnel the bulk sample A of seeds into separate chutes 53that are sized to accommodate a particular volume for processing by avision system 54. The seeds from each chute 53 fall by force of gravitypast the vision system 54. In the exemplary embodiment, the visionsystem 54 comprises an illuminating device, at least one sensing device,and a controller configured to control the illuminating and sensingdevices.

In the exemplary embodiment, the illuminating device comprises a lightsource that uses a bulb configured to emit electromagnetic radiation ata wavelength spectrum that illuminates the RFP marker. In otherembodiments, the light source may be any light source that permits theimage sensing device to discern the RFP marker. As such, in variousembodiments the light source and the marker may be paired so as toincrease the ability of the vision system to discern the presence of themarker. The exemplary embodiment includes multiple CCD cameras withfilters (i.e. red band-pass filters) configured to enhance theillumination and to aid in discerning the presence of the RFP marker.Although other embodiments may use fewer cameras, the exemplaryembodiment allows seeds falling past the vision system 54 to be viewedfrom the front and back. In other embodiments, any vision system 54configured to discern the presence of a RFP marker may be used,including, but not limited to, CCD devices, CMOS devices and othervision sensors.

Step 16 of FIG. 1 relates to sorting the seeds containing a geneticelement of interest based on the presence or absence of the marker. Thesorting function is carried out by a sorting device. In the exemplaryembodiment shown in FIG. 2, the sorting device 55 comprises a number ofindividual pneumatic ejectors that emit a controlled blast of air (suchas an “air knife” for example) configured for sorting seeds that exhibitthe RFP marker as the seeds pass through the sorting device. Seedsexhibiting the RFP marker are sorted into containers 56, identified inthe figure with a “+” symbol. Seeds that do not contain the marker fallinto containers 58, identified in the figure with a “−” symbol. Althoughnot shown in the figure, in other embodiments the seeds contained in the“−” container 58 may be re-routed through the hopper 52 so that theseseeds make a successive pass through the system 50. In such a manner anyseeds that were not identified as exhibiting the marker may beidentified in one or more successive passes through the system 50.

The above described method allows the processing of a large quantity ofseeds, a portion of which include a marker that is associated with agenetically transformed seed including a desired trait. However in otherembodiments, a bulk sample may include various seeds having differentmarkers associated with different desired traits, or seeds that includemore than one marker associated with different desired traits. Althoughthe exemplary embodiment described above may accommodate thesesituations, in other instances it may be advantageous to evaluate seedson a seed-by-seed basis. As a result, in various embodiments, thepresent invention contemplates singulating seeds from a bulk sampleprior to evaluating the seeds for the presence or absence of a marker ora group of markers associated with a desired trait or group of traits.

FIG. 3 shows a system 100 employing the above method in accordance withone embodiment of the present invention. The system 100 shown in thefigure includes an exemplary embodiment of a singulating devicecomprising a plurality of elongate hollow structures operativelyconnected to a vacuum source used to singulate seeds from a bulk sample.Specifically, the system 100 of the exemplary embodiment includes ahopper 102 into which a bulk sample A of seeds may be placed. A rotatingdrum 104 is positioned adjacent the hopper 102. The rotating drum 104includes a plurality of elongate hollow structures 106 that extend intoa substantially hollow interior of the drum 104, which is connected to avacuum source (not shown). The vacuum source creates a zone of negativepressure within the interior of the drum 104, and as a result, distalends of the elongate hollow structures 106 create discrete areas ofnegative pressure. As shown in the figure, the drum 104 rotates adjacentan open end of the hopper 102. The hopper 102 is configured such thatseeds are urged toward the open end and adjacent the rotating drum 104.The drum 104 rotates in the direction shown such that the elongatehollow structures 106 pass through a portion of the seeds of the bulksample A adjacent the drum 104. As the elongate hollow structures 106rotate through the bulk sample A, the discrete areas of negativepressure located at the distal ends of the elongate hollow structures106 attract individual seeds 101. The size of the distal ends of each ofthe elongate hollow structures 106 is configured such the discrete areaof negative pressure at the distal end of each of the hollow structures106 attracts a single seed 101. As shown in the figure, as the elongatehollow structures 106 rotate through the open end of the hopper 102,individual seeds 101 attach to the distal ends of the elongate hollowstructures 106. As the drum 104 continues to rotate in the directionshown, the seeds 101 travel toward a conveyor belt 110. Rotating drivingmembers 112 drive the conveyor belt 110 in the direction shown. Invarious embodiments, the speed of the driving members 112 may beadjusted to optimize downstream evaluating and sorting of the seeds 101.

At a position adjacent the conveyor belt 110, a deflector plate 108 (oralternatively, in some embodiments, a compressed air source configuredfor detaching individual seeds from the elongate hollow structures 106)deflects the seeds 101 from the distal ends of the elongate hollowstructures 106 and onto the conveyor belt 110. In the exemplaryembodiment, the deflector plate 108 contacts between the distal ends ofthe elongate hollow structures 106 and the seeds 101 carried by them sothat the area of negative pressure is temporarily blocked, effectivelyreleasing the seeds 101 from the distal ends of the elongate hollowstructures 106. Thus, the seeds 101 are singulated along the conveyorbelt 110. In various embodiments, the rotating drum 104 may have asingle radial pattern of elongate hollow structures 106, or, as shown inthe figure, it may have a series of elongate hollow structures 106arranged in a radial pattern.

A vision system 114 and sorting system 115 are shown downstream from therotating drum. As noted above, the vision system 114 comprises anilluminating device, at least one sensing device, and a controllerconfigured to control the illuminating and sensing devices. In theexemplary embodiment, the illuminating device comprises a light sourcethat uses a bulb configured to emit electromagnetic radiation at awavelength spectrum matched to illuminate the fluorescent proteinmarker. The exemplary embodiment includes multiple CCD cameras andfilters configured to enhance the illumination and to aid in discerningthe presence of the marker. The seeds 101 are sorted by the sortingsystem into “+” containers 116 and “−” containers 118. In the exemplaryembodiment, a series of mechanical deflectors and/or valves (see element55, FIG. 8, for example) are used to sort the pattern of singulatedseeds based on the presence or absence of the marker. The seedscontaining the marker are sorted into the “+” containers 116; the seedsthat do not contain the marker are sorted into the “−” containers 118.Although not shown in the figure, in other embodiments the seedscontained in the “−” container 118 may be re-routed through the hopper102 so that these seeds make a successive pass through the system 100.In such a manner any seeds that were not identified as exhibiting themarker may be identified in one or more successive passes through thesystem 100.

FIG. 4 shows another exemplary embodiment of a singulating, evaluating,and sorting system 200 employing a method in accordance with oneembodiment of the present invention. In the exemplary embodiment, avibratory stepped bowl 202 is configured to singulate seeds from a bulksample A and pass the singulated seeds 201 through an exit aperture 206which may be located, for example, near an upper periphery of thestepped bowl 202 of the system 200. For example, in some embodiments,the system may include a commercially-available seed counter (such asthe Seedburo 801 Count-A-Pak™ vibratory counter device manufactured bySeedburo Equipment Company in Chicago, Ill.). In such embodiments, theseeds 201 of the bulk sample A may be loaded into the stepped bowl 202of the system 200 such that as the stepped bowl 202 is vibrated, theseeds 201 may be lined up in single file along a periphery of the tracks204 defined on the steps of the stepped bowl 202 and advanced toward theexit aperture 206 and out through an exit chute 207 onto a conveyor belt210. As noted above, the conveyor belt 210 is driven by rotating drivingmembers 212 that drive the conveyor belt 210 in the direction shown. Invarious embodiments, the speed of the driving members 212 may beadjusted to optimize downstream evaluating and sorting of the seeds 201.

A vision system 214 and sorting system 215 are shown downstream from thebowl 202. As noted above, the vision system 214 comprises anilluminating device, at least one sensing device, and a controllerconfigured to control the illuminating and sensing devices. In theexemplary embodiment, the illuminating device comprises a light sourcethat uses a bulb configured to emit electromagnetic radiation at awavelength spectrum matched to illuminate the fluorescent proteinmarker. One exemplary embodiment includes multiple CCD cameras andfilters configured to enhance the illumination and to aid in discerningthe presence of the marker. The seeds 201 are sorted by the sortingsystem 215 into “+” containers 216 and “−” containers 218. In theexemplary embodiment, a series of mechanical deflectors are used to thesingulated seeds 201 based on the presence or absence of the marker. Inthe exemplary embodiment, seeds containing the marker are sorted intothe “+” containers 216 and seeds that do not contain the marker aresorted into the “−” containers 218. Although not shown in the figure, inother embodiments the seeds 201 contained in the “−” container 218 maybe re-routed through the hopper 202 so that these seeds make asuccessive pass through the system 200. In such a manner any seeds thatwere not identified as exhibiting the marker may be identified in one ormore successive passes through the system 200.

FIG. 5 shows another exemplary system 300 for singulating, evaluating,and sorting seeds based on the presence or absence of a marker inaccordance with an embodiment of the present invention. The exemplaryembodiment includes a hopper 302 that receives a bulk sample A of seedsand feeds the seeds onto a disk 304. In the exemplary embodiment, thehopper 302 includes a gate 303 that allows a controlled release of aquantity of seeds 301 onto the disk 304 based on gravitational force.The disk 304 is operatively connected to a motor (not shown) through ashaft 305 such that the disk 304 rotates in the direction shown. Thedisk 304 also includes a series of seed gates 308 that are located alonga periphery 306 of the disk 304. The seed gates 308 are configured toreceive individual seeds 301 that have been forced toward the periphery306 of the disk 304 due to centrifugal force caused by the rotation ofthe disk 304. In the exemplary embodiment, the gates 308 arecontrollable such that each gate 308 may open to release individualseeds 301. An exit chute 307 is located adjacent the periphery 306 ofthe disk 304. In the exemplary embodiment, each gate 308 may beindividually controlled to open and release an individual seed 301 whenthe gate 308 is adjacent the exit chute 307. A seed 301 released by agate 308 when adjacent the exit chute 307 travels through the exit chute307 and is deposited onto a conveyor belt 310. As noted above, theconveyor belt 310 is driven by rotating driving members 312 that drivethe conveyor belt 310 in the direction shown. In various embodiments,the speed of the driving members 312 may be adjusted to optimizedownstream evaluating and sorting of the seeds 301. As a result, seeds301 are singulated along the conveyor belt 310. As similarly describedabove, a vision system 314 and sorting system 315 are shown downstreamfrom the rotating disk 304 for evaluating and sorting seeds 301 based onthe presence or absence of the marker.

FIG. 6 shows yet another exemplary system 400 for singulating,evaluating, and sorting seeds based on the presence or absence of themarker in accordance with an exemplary embodiment of the presentinvention. In the exemplary embodiment, a hopper 402 is configured toreceive a bulk sample A of seeds. The hopper 402 may be vibrating andmay include a forward pitch that leads to an opening 403 such that seedsfrom the bulk sample A leave the opening 403 at a controlled rate. Asloped device 404 is located adjacent the opening 403 of the hopper 402.The sloped device include a first end 405 that is located adjacent theopening 403 of the hopper 402, and a second end 407 that is located alower elevation than the first end 405. The sloped device of theexemplary embodiment includes at least one groove 406 configured to forma channel that aligns seeds exiting the hopper 402 into a single filerow of individual seeds 401. In the exemplary embodiment, the groove 406is v-shaped, however in other embodiments the groove 406 may be anyother shape, such as u-shaped, that is configured to align seeds into asingle file row of individual seeds 401. Seeds 401 aligned in the groove406 of the sloped device 404 are urged toward the second end 407 of thesloped device 404 by gravitational force. A conveyor belt 410 is locatedbelow the second end 407 of the sloped device 404 to receive individualseeds 401 that fall by force of gravity from the second end 407 of thesloped device 404. As noted above, the conveyor belt 410 includesrotating driving members 412 that drive the conveyor belt 410 in thedirection shown. In various embodiments, the speed of the drivingmembers 412 may be adjusted to optimize downstream evaluating andsorting of the seeds 401. As a result, seeds 401 are singulated alongthe conveyor belt 410. As similarly described above, although not shown,a vision system and sorting system (see FIG. 8, for example) are locateddownstream from the sloped device for evaluating and sorting seeds basedon the presence or absence of the marker. As described in theExperimental Example herein, a vibratory hopper 402 singulation device,coupled with a Satake ScanMaster® sorting system may result inrelatively efficient and accurate sorting results. For example, usingsuch an exemplary system, the sorting step 16 may comprises sorting theseeds at a speed of at least about 500 seeds per minute, and in someembodiments at a speed of at least about 750 seeds per minute.

The foregoing merely illustrates how exemplary embodiments of thepresent invention distinguish seeds containing a genetic element ofinterest from a bulk sample. Referring now to FIG. 7, a block diagram ofan exemplary electronic device (e.g., PC, laptop, PDA, etc.) configuredto execute the method of distinguishing seeds containing a geneticelement of interest of exemplary embodiments of the present invention isshown. The electronic device may include various means for performingone or more functions in accordance with exemplary embodiments of thepresent invention, including those more particularly shown and describedherein. It should be understood, however, that the electronic device mayinclude alternative means for performing one or more like functions,without departing from the spirit and scope of the present invention. Asshown, the electronic device may generally include means, such as aprocessor, controller, or the like 740 connected to a memory 742, forperforming or controlling the various functions of the entity.

The memory can comprise volatile and/or non-volatile memory, andtypically stores content, data or the like. For example, the memorytypically stores content transmitted from, and/or received by, theelectronic device. Also for example, the memory typically storessoftware applications, instructions or the like for the processor toperform steps associated with operation of the electronic device inaccordance with embodiments of the present invention. In particular, thememory 742 may store computer program code for an application and othercomputer programs. For example, in one exemplary embodiment of thepresent invention, the memory may store computer program code for, amongother things, evaluating at least some of the seeds of a bulk sample forthe presence or absence of a marker associated with at least some seedscontaining a genetic element of interest of the bulk sample using anevaluating device, and sorting the seeds containing a genetic element ofinterest based on the presence or absence of the marker.

In addition to the memory 742, the processor 740 can also be connectedto at least one interface or other means for displaying, transmittingand/or receiving data, content or the like. In this regard, theinterface(s) can include at least one communication interface 746 orother means for transmitting and/or receiving data, content or the like,as well as at least one user interface that can include a display 748and/or a user input interface 750. The user input interface, in turn,can comprise any of a number of devices allowing the electronic deviceto receive data from a user, such as a keypad, a touch display, ajoystick or other input device.

As described above and as will be appreciated by one skilled in the art,embodiments of the present invention may be configured as a method andapparatus. Accordingly, embodiments of the present invention may becomprised of various means including entirely of hardware, entirely ofsoftware, or any combination of software and hardware. Furthermore,embodiments of the present invention may take the form of a computerprogram product consisting of a computer-readable storage medium (e.g.,the memory 742 of FIG. 16) and computer-readable program instructions(e.g., computer software) stored in the storage medium. Any suitablecomputer-readable storage medium may be utilized including hard disks,CD-ROMs, optical storage devices, or magnetic storage devices.

Exemplary embodiments of the present invention have been described abovewith reference to block diagrams and flowchart illustrations of methods,apparatuses (i.e., systems) and computer program products. It will beunderstood that each block of the block diagrams and flowchartillustrations, and combinations of blocks in the block diagrams andflowchart illustrations, respectively, can be implemented by variousmeans including computer program instructions. These computer programinstructions may be loaded onto a general purpose computer, specialpurpose computer, or other programmable data processing apparatus toproduce a machine, such that the instructions which execute on thecomputer or other programmable data processing apparatus create a meansfor implementing the functions specified in the flowchart block orblocks.

These computer program instructions may also be stored in acomputer-readable memory that can direct a computer or otherprogrammable data processing apparatus to function in a particularmanner, such that the instructions stored in the computer-readablememory produce an article of manufacture including computer-readableinstructions for implementing the function specified in the flowchartblock or blocks. The computer program instructions may also be loadedonto a computer or other programmable data processing apparatus to causea series of operational steps to be performed on the computer or otherprogrammable apparatus to produce a computer-implemented process suchthat the instructions that execute on the computer or other programmableapparatus provide steps for implementing the functions specified in theflowchart block or blocks.

Accordingly, blocks of the block diagrams and flowchart illustrationssupport combinations of means for performing the specified functions,combinations of steps for performing the specified functions and programinstruction means for performing the specified functions. It will alsobe understood that each block of the block diagrams and flowchartillustrations, and combinations of blocks in the block diagrams andflowchart illustrations, can be implemented by special purposehardware-based computer systems that perform the specified functions orsteps, or combinations of special purpose hardware and computerinstructions.

The following Experimental Example is provided only by way of example,and not by way of limitation.

Experimental Example

Introduction

Fluorescent protein (FP) marked seed are being produced for use insterility systems and for evaluating the impact of one or more insertedgenes on yield. It is a laborious process to separate a segregatingpopulation of seed into those containing (+) and those not containing(−) a genetic element of interest.

A project was initiated to develop an automated sorting routine that maybe capable of efficiently separating FP+ from FP− seed using a vibratoryfeeder similar to that shown in FIG. 6

Results

A vibratory feeder (see FIG. 6, for example) was evaluated with theSatake ScanMaster® system to assess sorting speed and accuracy in someembodiments of the present invention. It is known that it takesapproximately 30 minutes to sort 3500 kernels by hand. The FP-discerningScanMaster® (see FIG. 8, for example) equipped with a vibratory feeder(see FIG. 6, for example) for singulation, was shown to be capable ofsorting the same sample in about 2 to 3 minutes. Thus, the substantiallyautomated method embodiments of the present invention reduced sortingtime by approximately 90%. Unfortunately, the ScanMaster® color sorterwas only able to remove 90 to 95% of the FP seed from a 50:50 mixture ina single pass. During this process a significant number of FP− seed werecomingled with FP+ seed in what should be a pure FP+ sample. This levelof performance then suggests that some manual sorting may be required insome method embodiments to achieve a desired level of purity.

For example, if a typical yield trial sample contained 4000 kernels (50%FP− and 50% FP+) it would require about 40 minutes to sort the FP seed.This is a sorting rate of 50 seeds/minute. If the same sample weresorted using substantially automated embodiments of the presentinvention, it would require less than 30 seconds to generate a 95% puresample of FP+ seed. For a 2000 kernel FP+ sample, there would be about100 FP− kernels that would need to be manually removed. If the manualsorting rate is 50 seeds/minute, the actual time to achieve a nearly100% pure sample of FP+ seed will be about 2 minutes. Furthermore, if asample is manually sorted while another is being sorted using the FPSorter the actual sorting time for a sample would be about 2 minutes.This results in a fifteen-fold reduction in time required for separatingFP+ seed from FP− seed.

If an automatic re-sort function (i.e. re-routing the sample backthrough the sorting procedure for one or more additional passes) isused, it may be possible to recover 99% of the FP+ seed from the initialsample without having to manually re-sort the FP− fraction. Such are-sort process extends the cycle time by 15 seconds for a 500 gram seedsample. Given that it will take 2 minutes to sort the FP+ fraction itmay be advantageous to routinely re-sort samples, especially small ones,to recover as much of the FP+ seed as possible.

As noted above, the expression of fluorescent proteins may benon-uniform across the surface area of seeds. As a result, the presentinvention also provides a method for distinguishing seeds containing amarker associated with genetic element of interest from a bulk sample byusing an electromagnetic energy emitting device that is configured toexcite a majority of the surface area of the seeds and/or by using anevaluating device that is configured receive emissions from a majorityof the surface area of the seeds. FIG. 9 shows a non-limiting schematicof a system incorporating a vacuum drum singulating device, anelectromagnetic energy emitting device, and an evaluating device inaccordance with one embodiment of the present invention. Specifically,FIG. 9 shows a system 500 that includes an exemplary embodiment of asingulating device comprising a plurality of elongate hollow structuresoperatively connected to a vacuum source used to singulate seeds from abulk sample. Specifically, the system 500 of the exemplary embodimentincludes a hopper 502 into which a bulk sample A of seeds may be placed.A rotating drum 504 is positioned adjacent the hopper 502. The rotatingdrum 504 includes a plurality of elongate hollow structures 506 thatextend into a substantially hollow interior of the drum 504, which isconnected to a vacuum source (not shown). The vacuum source creates azone of negative pressure within the interior of the drum 504, and as aresult, distal ends of the elongate hollow structures 506 creatediscrete areas of negative pressure. As shown in the figure, the drum504 rotates adjacent an open end of the hopper 502. The hopper 502 isconfigured such that seeds are urged toward the open end and adjacentthe rotating drum 504. The drum 504 rotates in the direction shown suchthat the elongate hollow structures 506 pass through a portion of theseeds of the bulk sample A adjacent the drum 504.

As the elongate hollow structures 506 rotate through the bulk sample A,the discrete areas of negative pressure located at the distal ends ofthe elongate hollow structures 506 attract individual seeds 501. Thesize of the distal ends of each of the elongate hollow structures 506 isconfigured such the discrete area of negative pressure at the distal endof each of the hollow structures 506 attracts a single seed 501. Asshown in the figure, as the elongate hollow structures 506 rotatethrough the open end of the hopper 502, individual seeds 501 attach tothe distal ends of the elongate hollow structures 506. As the drum 504continues to rotate in the direction shown, a deflector plate 508 (oralternatively, in some embodiments, a compressed air source configuredfor detaching individual seeds from the elongate hollow structures 506)deflects the seeds 501 from the distal ends of the elongate hollowstructures 506 and onto the conveyor belt 510. In the exemplaryembodiment, the deflector plate 508 contacts between the distal ends ofthe elongate hollow structures 506 and the seeds 501 carried by them sothat the area of negative pressure is temporarily blocked, effectivelyreleasing the seeds 501 from the distal ends of the elongate hollowstructures 506.

As shown generally in FIG. 9, the system 500 may also include aspecialized vision system 554 which, in some embodiments, may comprise:(1) an electromagnetic energy emitting device, such as an illuminatingdevice 554 a that emits electromagnetic energy at a particular frequencychosen to illuminate and thereby “excite” a marker, and (2) anevaluating device, such as an image sensing device 554 c that maydiscern seeds expressing the marker. In various embodiments, the presentinvention may include an electronmagnetic energy emitting device that isconfigured to excite a majority of the surface area of the seeds and/oran evaluating device that is configured to receive emissions from amajority of the surface area of the seeds. In the depicted embodiment,the system 500 includes a pair of illuminating devices 554 ccollectively configured to excite a majority of the surface area of theseeds. In such a manner, the image sensing device 554 c may evaluate amajority of the surface area of the seeds to determine the presence orabsence of a marker.

In the depicted embodiment, image sensing device 554 c may be furtherconfigured for translating the emission into substantially “white”light. Thus, the image sensing device may assign substantially binaryvalues to each seed based on the presence or absence of the markerwherein seeds containing a genetic element of interest (tagged with therelatively “bright” marker) are marked “positive” (and thereby deflectedand/or other wise directed into one or more “+” containers 556. Seedsthat do not contain a genetic element of interest, or other particulatedebris (which may be translated into a “dark” or “negative” result) maybe dropped and/or otherwise directed into one or more “−” containers558. As shown in FIG. 9, the image sensing device 554 c (or othercomponent of a vision system 554 may be in communication with thedeflector plate 508, which may also include a guide plate 565 that mayrotate for directing the “positive” (i.e. seeds containing a geneticelement of interest (which the image sensing device 554 c may detect as“white” or “bright” seeds)) into one or more “+” containers 556 inresponse to a binary positive or “1” signal received from the imagesensing device 554 c or other processing component of the vision system554. The guide plate 565 may also be configured for directing the“negative” (i.e. seeds that do not contain a genetic element of interestor particulate debris (which the image sensing device 554 c may detectas “dark” seeds)) into one or more “−” containers 558 in response to abinary negative or “0” signal received from the image sensing device 554c or other processing component of the vision system 554.

Many different configurations are possible for distinguishing seedscontaining a marker associated with genetic element of interest from abulk sample by using an electromagnetic energy emitting device that isconfigured to excite a majority of the surface area of the seeds and/orby using an evaluating device that is configured receive emissions froma majority of the surface area of the seeds. Another non-limitingexample is shown generally in FIG. 10, which depicts a sorting system600 that includes a specialized vision system 654 which, in someembodiments, may comprise: (1) an electromagnetic energy emittingdevice, such as an illuminating device 654 a, that emits electromagneticenergy at a particular frequency chosen to illuminate and thereby“excite” a marker, and (2) an evaluating device, such as an imagesensing device 654 c that may discern seeds expressing the marker. Inthe depicted embodiment, the system 600 includes a pair of illuminatingdevices 654 a configured to collectively excite a majority of thesurface area of the seeds and a pair of image sensing devices 654 cconfigured to collectively receive emissions from a majority of thesurface area of the seeds. In such a manner, the image sensing device654 c may evaluate a majority of the surface area of the seeds todetermine the presence or absence of a marker.

As similarly described above, the evaluating device 654 c of FIG. 10 (orother component of a vision system 654) may be in communication with asorting device 655 (which may comprise, for example, a valve deviceand/or compressed air jet device) configured for directing the“positive” (i.e. seeds containing a genetic element of interest (whichthe image sensing device 654 c may detect as “white” or “bright” seeds))into one or more “+” containers 656 in response to a binary positive or“1” signal received from the image sensing device 654 c or otherprocessing component of the vision system 654. The sorting device 655may also be configured for directing the “negative” (i.e. seeds that donot contain a genetic element of interest or particulate debris (whichthe image sensing device 654 c may detect as “dark” seeds)) into one ormore “−” containers 658 in response to a binary negative or “0” signalreceived from the image sensing device 654 c or other processingcomponent of the vision system 654.

Many modifications and other embodiments of the invention set forthherein will come to mind to one skilled in the art to which thisinvention pertains having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

What is claimed is:
 1. A method for distinguishing seeds containing agenetic element of interest from a bulk sample, the method comprising:associating a red fluorescent protein marker with at least some of theseeds of the bulk sample; evaluating at least some of the seeds of thebulk sample for the presence or absence of the red fluorescent proteinmarker using a seed color sorter; and sorting the seeds containing agenetic element of interest based on the presence or absence of the redfluorescent protein marker; wherein the sorting step comprisesdispensing the sorted individual seeds into at least one container, thecontainer including seeds having the red fluorescent protein markerpresent or seeds in which the red fluorescent protein marker is absent,and wherein individual seeds are singulated from the bulk sample using asingulating device prior to said evaluating at least some of the seedsof the bulk sample for the presence or absence of the red fluorescentprotein marker, and further comprising associating a plurality ofsupplemental markers with at least some of the seeds containing acorresponding plurality of additional genetic elements of interest ofthe bulk sample; evaluating at least one of the seeds of the bulk samplefor the presence or absence of the plurality of supplemental markersusing the seed color sorter; and sorting the seeds containing theplurality of additional genetic elements of interest based on thepresence or absence of the a plurality of supplemental markers.
 2. Themethod according to claim 1, wherein the plurality of supplementalmarkers are selected from the group consisting of: yellow fluorescentproteins; yellow/orange fluorescent proteins; orange fluorescentproteins; orange/red fluorescent proteins; red/orange fluorescentproteins; red fluorescent proteins; cyan fluorescent proteins; andcombinations thereof.
 3. The method according to claim 1, wherein thesorting step comprises sorting the seeds at a speed of at least about500 seeds per minute.
 4. The method according to claim 1, wherein thesorting step comprises sorting the seeds at a speed of at least about750 seeds per minute.
 5. The method according to claim 1, wherein thered fluorescent protein marker is discernable when excited by a certainenergy having a wavelength ranging from substantially about 500 nm tosubstantially about 580 nm, the certain energy having a peak atsubstantially about 550 nm.
 6. The method according to claim 1, whereinsaid evaluating step comprises: exciting the seeds containing a geneticelement of interest with the certain energy; and detecting an emissionresulting at least in part from the exciting step, the emission having awavelength ranging from substantially about 500 nm to substantiallyabout 600 nm, the emission having a peak at substantially about 580 nm.7. The method according to claim 1, wherein the singulating stepcomprises singulating individual seeds using a plurality of elongatehollow structures operatively connected to a vacuum source.
 8. Themethod according to claim 1, wherein the singulating step comprisesreceiving the bulk sample in a hopper and vibrating the hopper so as topropel individual seeds along an inclined spiral ramp and through a gapconfigured to permit the passage of one seed at a time.
 9. The methodaccording to claim 1, wherein the singulating step comprises: receivingthe bulk sample in a hopper; feeding seeds from the hopper to arotatable disk; and rotating the rotatable disk so as to distribute theseeds to a periphery of the rotatable disk by application of centrifugalforce.
 10. The method according to claim 1, wherein the singulating stepcomprises: receiving the bulk sample in a hopper; vibrating the bulksample such that seeds tend to move towards an opening in the hopper;distributing seeds from the opening in the hopper to a sloped devicethat includes at least one groove such that a line of individual seedsis formed in the groove; and depositing individual seeds by gravityforce from the sloped device onto a conveying device.
 11. The method ofclaim 1, wherein the genetic element of interest comprises malesterility, and wherein the absence of the red fluorescent protein markerindicates male sterility.
 12. The method according to claim 1, whereinsaid evaluating step comprises evaluating seeds for the presence of amarker using at least one image sensing device configured todifferentiate between a range of normal seed emissions and an emissionfrom the red fluorescent protein marker.
 13. The method according toclaim 12, wherein the image sensing device is a CCD device or a CMOSdevice.
 14. The method according to claim 12, wherein the seed colorsorter further comprises at least one filter disposed substantiallybetween the image sensing device and the seeds containing a geneticelement of interest, the at least one filter configured for passing theemission from the red fluorescent protein marker to the image sensingdevice.
 15. The method according to claim 1, wherein said evaluatingstep comprises evaluating seeds for the presence of a marker using atleast one image sensing device configured to differentiate between arange of normal seed emissions and at least one emission from one ormore of the plurality of supplemental markers.
 16. The method accordingto claim 15, wherein the seed color sorter further comprises at leastone tunable filter disposed substantially between the image sensingdevice and the seeds containing a genetic element of interest, the atleast one tunable filter configured for selectively passing the at leastone emission from one or more of the plurality of supplemental markersto the image sensing device.
 17. A computer program product fordistinguishing seeds containing a genetic element of interest that havea red fluorescent protein marker associated with at least some of theseeds of a bulk sample, the computer program product comprising acomputer-readable storage medium having computer-readable program codeinstructions stored therein comprising: a first set of instructions forevaluating at least some of the seeds of the bulk sample for thepresence or absence of the red fluorescent protein marker using a seedcolor sorter; and a second set of instructions for sorting the seedscontaining a genetic element of interest based on the presence orabsence of the red fluorescent protein marker; wherein the second set ofinstructions comprises instructions for dispensing the sorted individualseeds into at least one container, the container including seeds havingthe red fluorescent protein marker present or seeds in which the redfluorescent protein marker is absent, and further comprising a third setof instructions for controlling a singulating device to singulateindividual seeds from the bulk sample prior to evaluating at least someof the seeds of the bulk sample for the presence or absence of the redfluorescent protein marker, and wherein at least a portion of the seedsof the bulk sample contain a plurality of additional genetic elements ofinterest associated with a corresponding plurality of supplementalmarkers, the computer program product further comprising: a fourth setof instructions for evaluating at least one of the seeds of the bulksample for the presence or absence of the plurality of supplementalmarkers using the seed color sorter; and a fifth set of instructions forsorting the seeds containing the plurality of additional geneticelements of interest based on the presence or absence of the a pluralityof supplemental markers.
 18. The computer program product according toclaim 17, wherein the plurality of supplemental markers are selectedfrom the group consisting of: yellow fluorescent proteins; yellow/orangefluorescent proteins; orange fluorescent proteins; orange/redfluorescent proteins; red/orange fluorescent proteins; red fluorescentproteins; cyan fluorescent proteins; and combinations thereof.
 19. Thecomputer program product according to claim 17, wherein the second setof computer instructions for sorting comprises sorting the seeds at aspeed of at least about 500 seeds per minute.
 20. The computer programproduct according to claim 17, wherein the second set of computerinstructions for sorting comprises sorting the seeds at a speed of atleast about 750 seeds per minute.
 21. The computer program productaccording to claim 17, wherein said third set of instructions comprisesa set of instructions for singulating individual seeds using a pluralityof elongate hollow structures operatively connected to a vacuum source.22. The computer program product according to claim 17, wherein saidthird set of instructions comprises a set of instructions for receivingthe bulk sample in a hopper and vibrating the hopper so as to propelindividual seeds along an inclined spiral ramp and through a gapconfigured to permit the passage of one seed at a time.
 23. The computerprogram product according to claim 17, wherein said third set ofinstructions comprises: a set of instructions for receiving the bulksample in a hopper, feeding seeds from the hopper to a rotatable disk,and rotating the rotatable disk so as to distribute the seeds to aperiphery of the rotatable disk by application of centrifugal force. 24.The computer program product according to claim 17, wherein said thirdset of instructions comprises: a set of instructions for receiving thebulk sample in a hopper, vibrating the bulk sample such that seeds tendto move towards an opening in the hopper, distributing seeds from theopening in the hopper to a sloped device that includes at least onegroove such that a line of individual seeds is formed in the groove, anddepositing individual seeds by gravity force from the sloped device ontoa conveying device.
 25. The method of claim 17, wherein the geneticelement of interest comprises male sterility, and wherein the absence ofthe red fluorescent protein marker indicates male sterility.
 26. Thecomputer program product according to claim 17, wherein the redfluorescent protein marker is discernable when excited by a certainenergy having a wavelength ranging from substantially about 500 nm tosubstantially about 580 nm, the certain energy having a peak atsubstantially about 550 nm.
 27. The computer program product accordingto claim 26, wherein said first set of instructions comprises: excitingthe seeds containing a genetic element of interest with the certainenergy; and detecting an emission resulting at least in part from theexciting step, the emission having a wavelength ranging fromsubstantially about 500 nm to substantially about 600 nm, the certainenergy having a peak at substantially about 580 nm.
 28. The computerprogram product according to claim 17, wherein said first set ofinstructions comprises evaluating seeds for the presence of a markerusing at least one image sensing device configured to differentiatebetween a range of normal seed emissions and an emission from the redfluorescent protein marker.
 29. The computer program product accordingto claim 28, wherein the image sensing device is a CCD device or a CMOSdevice.
 30. The computer program product according to claim 28, whereinthe seed color sorter further comprises at least one filter disposedsubstantially between the image sensing device and the seeds containinga genetic element of interest, the at least one filter configured forpassing the emission from the red fluorescent protein marker to theimage sensing device.
 31. The computer program product according toclaim 17, wherein said third set of instructions comprises evaluatingseeds for the presence of a marker using at least one image sensingdevice configured to differentiate between a range of normal seedemissions and at least one emission from one or more of the plurality ofsupplemental markers.
 32. The computer program product according toclaim 31, wherein the seed color sorter further comprises at least onetunable filter disposed substantially between the image sensing deviceand the seeds containing a genetic element of interest, the at least onetunable filter configured for selectively passing the at least oneemission from one or more of the plurality of supplemental markers tothe image sensing device.
 33. A method for distinguishing seedscontaining a genetic element of interest from a bulk sample, the methodcomprising: associating a marker with at least some of the seeds of thebulk sample; exciting the seeds using an electromagnetic energy emittingdevice; evaluating at least some of the seeds of the bulk sample for thepresence or absence of the marker using a seed color sorter; and sortingthe seeds containing a genetic element of interest based on the presenceor absence of the marker; wherein the sorting step comprises dispensingthe sorted individual seeds into at least one container, the containerincluding seeds having a red fluorescent protein marker present or seedsin which a red fluorescent protein marker is absent, wherein theelectromagnetic energy emitting device is configured to excite amajority of the surface area of the seeds, wherein individual seeds aresingulated from the bulk sample using a singulating device prior to saidevaluating at least some of the seeds of the bulk sample for thepresence or absence of the red fluorescent protein marker, and furthercomprising associating a plurality of supplemental markers with at leastsome of the seeds containing a corresponding plurality of additionalgenetic elements of interest of the bulk sample; evaluating at least oneof the seeds of the bulk sample for the presence or absence of theplurality of supplemental markers using the seed color sorter; andsorting the seeds containing the plurality of additional geneticelements of interest based on the presence or absence of the a pluralityof supplemental markers.
 34. The method of claim 33, wherein the geneticelement of interest comprises male sterility, and wherein the absence ofthe red fluorescent protein marker indicates male sterility.
 35. Themethod according to claim 33, wherein exciting the seeds using anelectromagnetic energy emitting device comprises exciting the seedsusing at least one illuminating device configured to illuminate amajority of the surface area of the seeds.
 36. The method according toclaim 35, wherein exciting the seeds using at least one illuminatingdevice further comprises using two or more illuminating devices arrangedto collectively illuminate a majority of the surface area of the seeds.37. The method according to claim 33, wherein evaluating at least someof the seeds of the bulk sample comprises evaluating at least some ofthe seeds of the bulk sample using at least one image sensing deviceconfigured to receive emissions from a majority of the surface area ofthe seeds.
 38. The method according to claim 37, wherein evaluating atleast some of the seeds of the bulk sample further comprises using twoor more image sensing devices arranged to collectively receive emissionsfrom a majority of the surface area of the seeds.
 39. The methodaccording to claim 33, further comprising: associating one or moresupplemental markers with at least some of the seeds containing acorresponding plurality of additional genetic elements of interest ofthe bulk sample; evaluating at least one of the seeds of the bulk samplefor the presence or absence of the supplemental markers using the seedcolor sorter; and sorting the seeds containing the plurality ofadditional genetic elements of interest based on the presence or absenceof the supplemental markers.
 40. The method according to claim 39,wherein the marker and the supplemental markers are selected from thegroup consisting of: green fluorescent proteins; green/yellowfluorescent proteins; yellow fluorescent proteins; yellow/orangefluorescent proteins; orange fluorescent proteins; orange/redfluorescent proteins; red/orange fluorescent proteins; red fluorescentproteins; cyan fluorescent proteins; and combinations thereof.
 41. Amethod for distinguishing seeds containing a genetic element of interestfrom a bulk sample, the method comprising: associating a red fluorescentprotein marker with at least some of the seeds of the bulk sample;evaluating at least some of the seeds of the bulk sample for thepresence or absence of the red fluorescent protein marker using a seedcolor sorter; and sorting the seeds containing a genetic element ofinterest based on the presence or absence of the red fluorescent proteinmarker, wherein the method further comprises re-routing the sorted seedsthrough the color seed sorter and re-evaluating the sorted seeds for thepresence or absence of the red fluorescent protein marker; whereinindividual seeds are singulated from the bulk sample using a singulatingdevice prior to said evaluating at least some of the seeds of the bulksample for the presence or absence of the red fluorescent proteinmarker, and further comprising associating a plurality of supplementalmarkers with at least some of the seeds containing a correspondingplurality of additional genetic elements of interest of the bulk sample;evaluating at least one of the seeds of the bulk sample for the presenceor absence of the plurality of supplemental markers using the seed colorsorter; and sorting the seeds containing the plurality of additionalgenetic elements of interest based on the presence or absence of the aplurality of supplemental markers.
 42. The method of claim 41 whereinre-routing the sorted seeds through the color seed sorter comprisesautomatically re-routing the sorted seeds through the color seed sorter.43. A method for distinguishing seeds containing a genetic element ofinterest from a bulk sample, the method comprising: associating a redfluorescent protein marker with at least some of the seeds of the bulksample; evaluating at least some of the seeds of the bulk sample for thepresence or absence of the red fluorescent protein marker using a seedcolor sorter; and sorting the seeds containing a genetic element ofinterest based on the presence or absence of the red fluorescent proteinmarker, wherein the sorting step comprises dispensing the sortedindividual seeds into at least one container, the container includingseeds having the red fluorescent protein marker present or seeds inwhich the red fluorescent protein marker is absent, wherein individualseeds are singulated from the bulk sample using a singulating deviceprior to said evaluating at least some of the seeds of the bulk samplefor the presence or absence of the red fluorescent protein marker, andwherein the evaluating step comprises exciting the seeds containing agenetic element of interest with a certain energy, detecting an emissionresulting at least in part from the exciting step, and translating theemission into substantially white light.